Project Summary/Abstract Accumulation of irreversibly arrested, senescent cells underlies numerous aging-associated pathologies. While rejuvenation of aged cells or clearance of senescent cells may beneficially impact aging, no current therapies exist. A key problem in the field of aging biology centers on understanding why cells adopt the growth-arrested, pro-survival state characteristic of senescent cells. A more thorough understanding of the alterations in cell-cycle biology that occur as a result of the aging process will open new avenues for therapeutic intervention. The Meyer lab specializes in performing single-cell analysis of live-cell imaging data. The lab employs recently developed fluorescent biosensors that enable the precise activity measurements of key cell-cycle regulatory proteins (e.g., CDK2, Ras, Erk, Akt) in individual live cells. Using this approach, the activity of several proteins within each individual cell can be measured, enabling multiple signaling networks to be compared quantitatively and temporally. This research program therefore proposes to apply live-cell, single-cell analysis technology to the study of aging-associated changes in cellular signaling dynamics. The goal of this proposal is to compare the dynamics of both Ras pathway signaling and the replication stress response between young and old in vivo aged cells to determine how aging affects these pathways. My central hypothesis is that aging increases replication stress in aged cells and alters the relative strength of the Erk and Akt signaling pathways downstream of Ras, ultimately resulting in impaired cellular proliferation. I plan to test this hypothesis with the following specific aims: 1. Identify aging-associated changes in downstream Ras signaling 2. Identify the impact of aging on replication stress response signaling and S-phase dynamics Successful completion of these aims will determine to what extent alterations in Ras downstream signaling and the replication stress response promote survival and impair cellular proliferation during aging. Understanding the aging-associated changes in these central signaling pathways will enable future work to identify upstream aging- induced changes in cellular machinery that play a causative role in the impairment of proliferation. Ultimately, this will facilitate the targeting of aging-specific changes in cell-cycle biology that represent promising targets for therapeutic reversal of the aging process.